| ObjectiveThe objective is to observe the impact of CUMS rats kidney-warming therapy on AC-cAMP-PKA signal pathway.The pathological changes and treatment effects of depression model rats are decided by observing cerebral tissues under light microscope to explore the mechanism of action so as to provide basis for clinical application.MethodSelected 84 male SD rats at the similar weight(200±20g)and score of Open-Field method and divided them into two parts,of which they were divided into 4 groups randomly to be normal control group,depression model control group,kidney-warming therapy group and fluoxetine hydrochloride group respectively.At present,CUMS that is the most recognized method in the world was used to build rat depression model.The author have arranged 21 days,and every 2 stimulations should not appear.After 7 different stimulations appeared alternately for 21 days,open-field test was used to evaluate to estimate whether the modeling was successful.In addition,intragastric administration was adopted.Since day 22,the corresponding medicines were given to kidney-warming therapy group and fluoxetine hydrochloride group by intragastric administration.Equivalent normal saline was given to the normal control group and depression model control group once a day for 21 days continuously.Hippocampus was obtained from 60 rats in the first part,and Elisa was used to determine the contents of AC and cAMP.Western blotting was used to test the PKAc protein level of hippocampus.Brain tissues were obtained from 24 rats in the second part.After the procedures of dehydration,pruning,embedding,HE staining,staining and microscopic examination,specimens were divided to take pictures and put into hard disk to record date.Result1.Open-field experimental result: except the normal control group,for the open-field experimental result aimed at 58 rats of the first part,the rats of the rest groups showed different degrees of depression changes after 21 days of CUMS stimulation.In the open-field experiment,after modeling in the model groups,the horizontal score and vertical score were markedly reduced(P<0.01),indicating that CUMS stimulation declined the athletic ability and exploring ability of rats,and the modeling was successful.After intragastric administration for 21 days,the depressive behaviors of rats in kidney-warming therapy group and fluoxetine hydrochloride group were improved markedly.In the open-field experiment,the horizontal score and vertical score of rats in kidney-warming therapy group and fluoxetine hydrochloride group were improved markedly.Compared with the depression model group,it had significant difference(P<0.01).For the comparison between kidney-warming therapy group and fluoxetine hydrochloride group,the behavioral index difference of both had no statistical difference(p >0.05),indicating that kidney-warming therapy could markedly improve the behaviors of rats,and the effect of anti-depression was close to fluoxetine hydrochloride.The open-field experimental result of 24 rats in the second part showed that the horizontal activity score and vertical activity score of model group,kidney-warming therapy group and fluoxetine hydrochloride group were markedly reduced compared with blank group after modeling was finished(day 21 and day 42),the difference had significant statistical significance(P<0.01).In day 21,the comparison between treatment groups and model group had no significant difference.In day 42,for the kidney-warming therapy group compared with model group,the horizontal activity score and vertical activity score were markedly increased,and the difference had statistical significance(P<0.05).For the fluoxetine hydrochloride group compared with model group,the horizontal activity score was markedly increased,and the difference had statistical significance(P<0.05),but the difference of vertical activity score had no statistical difference.The comparison of treatment groups had no statistical significance.For the comparison after drug administration(day 42)and modeling(21),the blank group and model group had no statistical difference.The horizontal activity score and vertical activity score of kidney-warming therapy group and fluoxetine hydrochloride group had significant statistical difference(P<0.01),indicating that kidney-warming therapy could effectively improve the depressive behaviors of rats,and the treatment effect had no significant difference from fluoxetine hydrochloride group.2.Result of ELISA detection on hippocampus AC and cAMP of rats: compared with blank group,AC content of hippocampus in model rat was markedly reduced(P<0.01).Compared with model group,AC content of hippocampus in fluoxetine hydrochloride group and kidney-warming therapy group was markedly increased(P<0.05,P<0.05).Compared with blank group,cAMP content of hippocampus in model rat was markedly reduced(P<0.01).Compared with model group,cAMP content of rat hippocampus in fluoxetine hydrochloride group and kidney-warming therapy group was markedly increased(P<0.05,P<0.05),indicating that AC and cAMP content of rat hippocampus in depression model was markedly reduced.The treatment of kidney-warming traditional Chinese medicine and fluoxetine hydrochloride could increase AC and cAMP content of rat hippocampus,which almost reached the level of blank group.3.Result of Western blotting detection on hippocampus PKAc of rats: compared with blank group,PKAc content of hippocampus in model rat was markedly reduced(P<0.01).Compared with model group,cAMP content of rat hippocampus in fluoxetine hydrochloride group and kidney-warming therapy group was markedly increased(P<0.01,P<0.01),indicating that kidney-warming therapy and fluoxetine hydrochloride could increase PKAc content of hippocampus in depression model rats.For the comparison between two groups,the difference had marked statistical significance(P<0.01).4.Pathological brain tissue examination result of depression model rats: compared with blank group,the model group had edema on the surface layer of cortex,pyramidal cells had mild and moderate edema,and brain tissue had light and moderate inflammatory cell infiltration.Compared with model group,the tissue structure of ectocinerea and white matter in kidney-warming therapy group was normal.The cell gradation of grey matter area was clear.Pyramidal cells and granule cells were in alignment.The size,quantity,morphology and distribution of cells were normal.Cell nucleus and plasma structure were clear.The staining was even and endochylema was light red.The cells were prominent and visible.The karyon was blue and clear.Pyramidal cells had mild edema.Mild inflammatory cell infiltration could be seen from the brain tissue.The tissue structure of ectocinerea and white matter area in fluoxetine hydrochloride group was normal.The cell gradation of grey matter area was clear.Pyramidal cells and granule cells were in alignment.The size,quantity,morphology and distribution of cells were normal.Cell nucleus and plasma structure were clear.The staining was even and endochylema was light red.The cells were prominent and visible.The karyon was blue and clear.Mild edema could be seen from pyramidal cells in a few specimens,and brain tissue had mild inflammatory cell infiltration.ConclusionThe anti-depression effect of kidney-warming therapy is clear,which could obviously improve the open-field experimental score of CUMS depression model rats and markedly improve behavior abnormity of depressive experimental rats.AC and cAMP of hippocampus were increased and PKAc expression of hippocampus was improved.AC-cAMP-PKA signal pathway was up-regulated.In addition,edema on the surface layer of cortex,pyramidal cell edema and inflammatory cell infiltration of brain tissue could be improved to have anti-depression effect and improve the depressive state of rats. |
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